METACYCLIC PROMASTIGOTES OF LEISHMANIA IN THE SALIVARY GLANDS OF EXPERIMENTALLY INFECTED PHLEBOTOMINE SANDFLIES

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1 Article available at or METACYCLIC PROMASTIGOTES OF LEISHMANIA IN THE SALIVARY GLANDS OF EXPERIMENTALLY INFECTED PHLEBOTOMINE SANDFLIES KILLICK-KENDRICK R.*, KILLICK-KENDRICK M.*, TANG Y.* & BASTIEN P.* Summary: Résumé : PROMASTIGOTES MÉTACYCLIQUES DE LEISHMANIA DANS LES Thirty one female Phlebotomus duboscqi experimentally infected GLANDES SALIVAIRES DE PHLÉBOTOMES INFECTÉS EN LABORATOIRE with Leishmania tropica were found with metacyclic promastigotes Des promastigotes métacycliques ont été observés dans les in their salivary glands. The presence of parasites in the glands glandes salivaires de 3 I Phlebotomus duboscqi femelles infectées was correlated with heavy infections of metacyclic promastigotes en laboratoire par Leishmania tropica. La présence de parasites in the stomodaeal valve and thoracic midgut of the fly and it is dans ces glandes est corrélée à des infestations importantes de suggested that the parasites may have migrated through the gut promastigotes métacycliques dans la valve stomodéale et la wall into the glands which lie against the valve in the thorax of the portion thoracique de l'intestin moyen du phlébotome, et il est fly. No evidence was found that gland invasion is an obligatory suggéré que les parasites peuvent avoir migré à travers la paroi stage of the life-cycle of Leishmania, but reports of wild-caught flies intestinale vers les glandes qui s'étendent le long de la valve with parasites in the glands, coupled with these laboratory thoracique du phlébotome. Aucune preuve n'a été apportée du observations, raise the possibility that regurgitation of parasites or caractère obligatoire d'une phase d'invasion des glandes dans le the migration of metacyclic promastigotes into the mouthparts may cycle des Leishmania; cependant, des observations de parasites not be the only mechanisms of transmission by bite. dans les glandes de phlébotomes capturés dans la nature, couplées à ces observations de laboratoire, nous font émettre KEY WORDS : Leishmania tropica, Phlebotomus duboscqi, life-cycle, l'hypothèse salivary que la régurgitation des parasites ou la migration de glands, transmission. promastigotes métacycliques vers les pièces buccales ne sont peutêtre pas les seuls mécanismes de transmission par piqûre. MOTS CLES : Leishmania tropica, Phlebotomus duboscqi, cycle évolutif, glandes salivaires, transmission. For the past sixty years, there has been much discussion on the precise mechanism by which a phlebotomine sandfly deposits an infective dose of leishmanial promastigotes into the skin of a vertebrate host. Reports of parasites in the proboscis of the fly (Adler & Theodor, 1929, 1930, 1935; Adler ET AL., 1938) suggested that invasion of the mouthparts was the culmination of the development in the alimentary tract of the vector, and that it was a prerequisite if the fly were to be able to pass on the infection (Killick-Kendrick, 1979). An alternative idea was based on observations that the pharynx and oesophagus of infected flies were sometimes blocked by a mass of parasites (Shortt ET AL., 1926). Influenced by the discovery of the transmission of plague by fleas with the alimentary canal blocked by YERSINIA PESTIS (see Parrot & Donatien, 1952), the notion arose that a plug of promastigotes in the foregut or anterior midgut of the sandfly was regurgitated into the skin of the invertebrate when an infected insect took a blood meal (Shortt & Swaminath, * Department of Biology, Imperial College at Silwood Park, Ascot, Berks SL5 7PY, UK. 1928; Napier, 1946). Adler and Theodor (1935) strongly opposed this idea on the grounds that the blockage was more apparent than real. They pointed out that the pharynx was highly expandable by the large muscles that enabled it to work as a pump, and presented a formidable case to support the idea that transmission was by small, highly active promastigotes in the mouthparts (reviewed by Killick-Kendrick, 1979, 1986). By 1985, Adler's theory had received strong support from reports of promastigotes in mouthparts in 18 combinations of sandflies and LEISHMANIA species (Killick- Kendrick, 1986). Doubts were then cast on his ideas when laboratory transmissions of LEISHMANIA were achieved in the apparent absence of parasites in the proboscis (Lainson ET AL, 1977; Pozio ET AL, 1985). After infected sandflies were shown to have difficulty in taking a bloodmeal (Killick-Kendrick ET AL, 1977b), it was suggested that parasites in the buccal cavity might interfere with the function of internal receptors thereby changing the feeding behaviour of the fly and enhancing the chances of parasites from the foregut being deposited in the skin of the vertebrate host (Killick- Kendrick & Molyneux, 1981). However, a detailed 55

2 KILLICK-KENDRICK R., KILLICK-KENDRICK M. TANG Y S BASTIEN P. study showed that, unlike other Diptera, phlebotomine sandflies seen to have no chemoreceptors in the cibarium (Jefferies, 1987), and it appears that this is not the explanation of multiple probing by infected flies. A new explanation of the changed feeding behaviour emerges from highly original observations by Schlein and colleagues who showed that promastigotes encased in the peritrophic membrane in the midgut of a sandfly produce chitinolytic enzymes that not only break down the membrane and permit the parasites to escape, but also damage the chitin of the stomodaeal valve (the cardia of some authors) (Schlein ET AL, 1991). Schlein ET AL. (1992) published electronmicrographs of a damaged valve and illustrated how they thought this could lead to its malfunction resulting in the deposition of promastigotes from the anterior midgut and foregut into the skin of a vertebrate as an infected fly attempted to feed. The action of the chitinolytic enzymes is inhibited by haemoglobin (Schlein & Jacobsen, 1994), which explains the absence of signs of damage in electronmicrographs of the cuticular intima of the valve or hindgut of several different species of flies infected with various parasites examined before, or shortly after, the digestion of the infecting blood meal. Schlein's ideas on the mechanism of transmission of LEISBMANIA by the bite of an infected sandfly are plausible and offer an explanation of multiple probing by infected flies and the difficulty they have in obtaining a full blood meal (Killick-Kendrick ET AL., 1977b, Beach ET AL., 1985). The possibility appears never to have been considered that another mode of transmission could sometimes be by promastigotes in the salivary glands being deposited in the skin of the vertebrate host with the saliva. In the present paper we describe such infections in experimentally infected sandflies and suggest that there is perhaps more than one mechanism of transmission by bite. MATERIALS AND METHODS Sandflies The flies were from a laboratory colony of PHLE BOTOMUS DUBOSCQI Neveu-Lemaire set up in June, 1988, with the eggs of females caught in CDC miniature light traps at Keur Moussa, Senegal. The methods for the initiation and maintenance of the colony are given by Killick-Kendrick & Killick-Kendrick (1987, 1991). The lines used originated from the progeny of individual females that were inbred and selected for their susceptibility to infections by LEISH MANIA TROPICA Wright. The observations reported in the present paper were made on the 13th to 24th generations after inbreeding was begun. Parasite The parasite was isolated in NNN medium from a cutaneous lesion of a patient in Kabul, Afghanistan, in June, It was typed at the Faculty of Medicine, Montpellier, the Istituto Superiore di Sanita, Rome, and the London School of Hygiene and Tropical Medicine and identified as L. TROPICA, zymodeme MON-58 (= LON- 20). The strain, No MHOM/AI788/KK27 (= LEM 1422 = ISS 423), was maintained in golden hamsters by the passage of amastigotes (Bastien & Killick-Kendrick, 1992). Infection of sandflies Washed suspensions of amastigotes were prepared from infected hamster skin tissue. The parasites were counted both in a haemocytometer and by the microbead method of Cenini ET AL. (1989). The proportion of viable amastigotes was estimated with fluorescent illumination after staining with ethidium bromide and fluorescein diacetate (Cenini ET AL, 1989), and the suspension was diluted with inactivated rabbit blood to give an estimated 3 x 10 5 viable amastigotes per 1.0 ml. The sandflies were permitted to feed on the suspension through a hamster cheek-pouch attached to a glass feeder warmed by circulating water (Bastien, 1990). Each fly with a full blood meal was estimated to have taken about 100 parasites. Maintenance and examination of infected flies On the day after the meal, fed flies were individually tubed, provided with a solution of sucrose (50:50 v/v) and kept at 25 C. They were examined at least twice a day until they had laid eggs when they were immediately dissected and examined. The head of each fly was cut off and an attempt was made to dissect out both salivary glands and mount them intact under a separate coverslip before the gut was removed. They were examined under phase contrast or interference illumination. Flies were not considered to have negative salivary glands unless both glands were examined. The intensity of infection in different parts of the alimentary tract was subjectively assessed and scored +++++, ++++, +++, ++ and +. RESULTS Promastigotes were seen in the salivary glands of 31 sandflies 9-21 days after infection. In 22 of these flies, only one of the two glands appeared to be infected. Three flies had promastigotes in both glands but, in six others, a gland was lost during dissection and only one was available for examination. The numbers of parasites seen were generally low. Of 34 infected glands, 21 had 1-5 parasites, 56 Parasite, 1996,.5, 55-60

3 METACYCLIC; PROMASTIGOTES OF Leishmania IN THE SALIVARY GLANDS Fig.l, 430X; Figs 2 & 4, 1180X; Fig. 3, 800X. Metacyclic promastigotes of Leishmania tropica in freshly dissected salivary glands of experimentally infected Phlebotomus duboscqi. Phase contrast illumination. 57

4 KILLICK-KENDRICK R., KILLICK-KENDRICK M., TANG Y. & BASTIEN P. five had 6-10, two had and six had > 20. T h e r e w e r e heavily infected, but a hamster inoculated with w a s a strong correlation b e t w e e n infected glands and the parasites did not develop a lesion. Eighteen months the intensity o f infection in the midgut, linked to the later, the strain was r e c o v e r e d b y culturing from the p r e s e n c e o f numerous metacyclic forms. Twenty four site o f inoculation and was typed b y Grimaldi ET AL. o f the flies with parasites in the glands had both hea ( ) as LE. NAIFFI Lainson & S h a w ; it is r e a s o n a b l e vily infected stomodaeal valves ( s c o r e d as or to suppose that the promastigotes in the salivary glands ++++) and thoracic midguts (++++). Eighteen o f the w e r e the same species. 24 had extraordinarily large numbers o f metacyclic pro- T h e r e are t w o routes by w h i c h promastigotes c o u l d mastigotes in these parts o f the alimentary tract. m o v e from the midgut to the salivary glands. Firstly, Lightly infected glands initially s e e m e d often to b e they may penetrate through negative. After long observation, the c o m m o n e s t first glands which lie pressed against the thoracic midgut indication o f infection was an intermittently moving fla- (Jobling, ). Most flies with parasites in the glands gellum. It was necessary to wait until the parasite had heavily infected stomodaeal valves and thoracic m o v e d revealing the body, and the parasite could midguts and, from electronmicrographs, promastigotes then b e s e e n pushing b e t w e e n the cells o f the gland. are k n o w n to burrow into the cells o f the anterior In glands with more than a d o z e n parasites, the pro- midgut ( M o l y n e u x ET AL., ; Killick-Kendrick ET AL, the gut wall into the mastigotes were visible without difficulty with phase 1977'A). Furthermore, Adler & T h e o d o r ( ) contrast or interference microscopy (but not with bright parasites throughout the h a e m o c o e l o m i c cavity o f t w o field). Cells o f o n e infected gland a p p e a r e d to have sandflies that had b e e n i m b e d d e d b e e n d a m a g e d b y the parasites (Fig. 3 ). All parasites in the glands l o o k e d like metacyclic promastigotes. T h e y w e r e small with a b o d y length o f only 5-10 um and a flagellum length at least twice that o f the body (Figs 1-4). N o n e was in division. It is not possible to estimate the true prevalence o f gland infections b e c a u s e it is certain that s o m e glands recorded as negative w e r e infected, but the parasites w e r e impossible to s e e. Nevertheless, no parasites w e r e s e e n in the glands o f many hundreds o f our experimentally infected flies and the overall prevalence was seemingly low. and found sectioned; curiously, they attached n o importance to their o b s e r vation. T h e s e c o n d, less likely, route is by the salivary duct w h i c h o p e n s towards the anterior tip o f the mouthparts (Jobling, ). This w o u l d b e likely only if the proboscis w e r e infected. Morphologically, the p r o m a s t i g o t e s in the salivary glands r e s e m b l e d parasites r e c o v e r e d from the pro boscis o f infected flies (Adler & T h e o d o r, , ; Killick-Kendrick, ). Their size and shape w e r e the s a m e as m e t a c y c l i c p r o m a s t i g o t e s, the form pre- adapted to life in the vertebrate host ( s e e review b y Killick-Kendrick, 1990), and it s e e m s p r o b a b l e they would b e infective and b e deposited in the skin during the act o f biting. Such a m e c h a n i s m o f transmission DISCUSSION O could b e advantageous in the evolution o f LEISHMANIA spp. since there is n o w much e v i d e n c e that sandfly ur observations are insufficient e v i d e n c e by saliva plays an important role enabling metacyclic pro themselves to c o n c l u d e that invasion o f the mastigotes to b e c o m e established in skin at the site o f salivary glands o f sandflies by promastigotes the bite (Titus & Ribeiro, ; Samuelson ET AL, ; is a normal part o f the life cycle o f LEISHMANIA. thermore, our w o r k w a s with a LEISHMANIA Fur T h e o d o s ET AL, ). species in Salivary glands are seldom examined and further obser v e c t o r. T h e r e are, vations on both natural and experimental infections however, t w o reports from Brazil o f promastigotes in must b e m a d e before the relevance o f promastigotes a fly w h i c h is not its natural the salivary glands o f wild-caught sandflies. Arias ET AL. in the glands to transmission can b e judged. Light infec ( ) found three female flies with infected glands tions are almost impossible to detect w h e n glands are among catches made in Amazonas State. T h e identity e x a m i n e d with bright field illumination, and it is n e c e s o f two o f the flies is not clear, but the third was a sary to use a X oil-immersion objective with either m e m b e r o f the SHANNONI group which could not b e phase contrast or interference illumination. Even with identified further b e c a u s e this group contains species this equipment, the glands have often to b e o b s e r v e d with isomorphic females. T h e parasite from the midgut for a long time before it is certain they are infected. o f this fly failed to infect a hamster, and its identity A further difficulty in seeing the promastigotes is that remains u n k n o w n. In the s e c o n d report, Naiff ET AL. they are o b s c u r e d b y d e g e r a t e cells and ( ) found promastigotes in the salivary glands o f w h i c h appear with progressive c h a n g e s o f the glands granules o n e o f two naturally infected s p e c i m e n s o f LU. SQUA- following a b l o o d meal (Adler & T h e o d o r, ). MIVENTRIS (Lutz & Neiva) caught at Balbina, Amazonas A review o f publications on transmission by bite leads State. T h e midgut and Malpighian tubules o f the fly to the conclusion that a search for a single m e c h a n i s m 58

5 METACYCUC PROMASTICOTES OF LELSHMAXIA IN THE SALIVARY GLANOS may be in vain. There is strong evidence that parasites may be regurgitated following damage to the stomodaeal valve by chitinolytic enzymes produced by the parasite (Schlein et al, 1991, 1992). It also seems highly probable that transmission could occur by the transfer of parasites from heavily infected mouthparts into the skin in the act of biting (Killick-Kendrick, 1979). Possibly invasion of the salivary glands and the deposition of metacyclic promastigotes into the skin with the saliva is a third way. ACKNOWLEDGEMENTS We are grateful to the Medical Research Council, London, and the Leverhulme Trust for financial support. The Afghan strain of L. tropica was isolated while R.K-K. was a Short Term Consultant for the WHO Eastern Mediterranean Regional Office. REFERENCES ADLER S. & THEODOR O. The mouth parts, alimentary tract, and salivary apparatus of the female in Phlebotomuspapatasi. Annals of Tropical Medicine and Parasitology, 1926, 20, ADLER S. & THEODOR O. Attempts to transmit Leishmania tropica by bite: the transmission of L. tropica by Phlebotomus sergenti. Annals of Tropical Medicine and Parasitology, 1929, 23, ADLER S. & THEODOR O. The exit of Leishmania infantum from the proboscis of Phlebotomus perniciosus. Nature, London, 1930, 126, 883- ADLER S. & THEODOR O. Investigations on Mediterranean kala azar. IX. Feeding experiments with Phlebotomus perniciosus and other species on animals infected with Leishmania infantum. Proceedings of the Royal Society, B, 1935, 116, ADLER S., THEODOR O. & WITENBERG G. Investigations on Mediterranean kala azar. XI. A study of leishmaniasis in Canea (Crete). Proceedings of the Royal Society, B, 1938, 125, ARIAS J.R. & DE FREITAS R.A. Sobre os vetores de leishmaniose cutanea na Amazonia central do Brasil. 2: Incidencia de flagelados em flebótomos selváticos. Acta Amazónica, 1978, BASTIEN P. Hamster cheek pouches compared with chick skins for membrane feeding of phlebotomine sandflies. Transactions of the Royal Society of Tropical Medicine and Hygiene, 1990, 84, 530. BASTIEN P. & KILLICK-KENDRICK R. Leishmania tropica infection in hamsters and a review of the animal pathogenicity of this species. Experimental Parasitology, 1992, 75, BEACH R., KILLU G. & LEEUWENBURG J. Modification of sand fly biting behavior by Leishmania leads to increased parasite transmission. American Journal of Tropical Medicine and Hygiene, 1985, 34, CENINI P., REEVE A.M. & NEAL R. TWO new techniques for quantitative determination of Leishmania amastigotes. Transactions of the Royal Society of Tropical Medicine and Hygiene, 1989, 83, GRIMALDI G., MOMEN H., NAIFF R.D., MCMAHON PRATT D. & BAR RETT T.V. Characterization and classification of leishmanial parasites from humans, wild mammals, and sand flies in the Amazon Region of Brazil. American Journal of Tropical Medicine and Hygiene, 1991, 44, JEFFERIES D. Labrocibarial sensilla in the female sandflylutzomyia longipalpis Lutz & Neiva (Diptera: Psychodidae). Canadian fournal of Zoology, 1987, 65, AAA-AAS. JOBLING B. Phlebotomus papatasi. In: Anatomical drawings of biting flies, British Museum (Natural History), London, 1987, KILLICK-KENDRICK M. & KILLICK-KENDRICK R. The initial establishment of sandfly colonies. Parassitologia, 1991, 33 (Supplì), KILLICK-KENDRICK R. Biology of Leishmania in phlebotomine sandflies. In: Biology of the Kinetoplastida (Lumsden, W.H.R. & Evans, D.A., eds). Volume 2, Academic Press, New York, 1979, KILLICK-KENDRICK R. The tranmission of leishmaniasis by the bite of the sandfly. Journal of the Royal Army Medical Corps, 1986, /32, KILLICK-KENDRICK R. The life-cycle of Leishmania in the sandfly with special reference to the form infective to the vertebrate host. Annales de Parasitologic humaine et comparée, 1990, 65, KILLICK-KENDRICK R. & KILLICK-KENDRICK M. The laboratory colonization of Phlebotomus ariasi (Diptera: Psychodidae). Annates de Parasitologic humaine et comparée, 1987, 52, KILLICK-KENDRICK R., LAINSON R., LEANEY A.J., WARD R.D. & SHAW J J. Promastigotes of L.b. braziliensis in the gut wall of Brazilian sandflies. Transactions of the Royal Society of Tropical Medicine and Hygiene, 1977, 71, 381. KILLICK-KENDRICK R., LEANEY A.J., READY P.D. & MOLYNEUX D.H. Leishmania in phlebotomid sandflies. IV. The transmission of Leishmania mexicana amazonensis to hamsters by the bite of experimentally infected Lutzomyia longipalpis. Proceedings of the Royal Society, B, 1977, 196, KILLICK-KENDRICK R. & MOLYNEUX D.H. Transmission of leishmaniasis by the bite of phlebotomine sandflies: possible mechanisms. Transactions of the Royal Society of Tropical Medicine and Hygiene, 1981, 75, LAINSON R., WARD R.D. & SHAW J.J. Experimental transmission of Leishmania chagasi, causative organism of Neotropical visceral leishmaniasis, by the sandfly Lutzomyia longipalpis. Nature, London, 1977, 266, MOLYNEUX D.H., KILLICK-KENDRICK R. & ASHFORD R.W. Leishmania in phlebotomine sandflies. III. The ultrastructure of Leishmania mexicana amazonensis in the midgut and pharynx of Lutzomyia longipalpis. Proceedings of the Royal Society, B, 1975, 190,

6 NAIFF R.D., FREITAS R.A., NAIFF M.F., ARIAS J.R., BARRETT T.V., MOMEN H. & GRIMALDI G. Epidemiological and nosological aspects of Leishmania naiffi Lainson & Shaw, Memorias do Instituto Oswaldo Cruz, 1991, 86, NAPIER L.E. Kala-azar. In: The Principles and Practice of Tropical Medicine, Macmillan, New York, 1946, PARROT L. & DONATIEN A. Autres observations sur l'infection naturelle des phlébotomes par la leishmaniose générale de l'homme et du chien en Algérie. Archives de l'institut Pasteur d'algérie, 1952, 30, Pozio E., MAROLI M., GRADONI L. & GRAMICCIA M. Laboratory transmission of Leishmania infantun to Rattus rattus by the bite of experimentally infected Phlebotomus pemiciosus. Transactions of the Royal Society of Tropical Medicine and Hygiene, 1985, 79, SAMUELSON J., LERNER E., TESH R. & TITUS R. A mouse model of Leishmania braziliensis infection produced by coinjection with sandflysaliva. Journal of Experimental Medicine, 1991, 173, SCHLEIN Y. & JACOBSEN R.L. Haemoglobin inhibits the development of infective promastigotes and chitinase secretion in Leishmania major cultures. Parasitology, 1994, 109, SCHLEIN Y., JACOBSEN R.L. & MESSER G. Leishmania infections damage the feeding mechanism of the sandfly vector and implement parasite transmission by bite. Proceedings of the National Academy of Science, USA, 1992, 89, SCHLEIN Y., JACOBSEN R.L. & SHLOMAI J. Chitinase secreted by Leishmania functions in the sandfly vector. Proceedings of the Royal Society, B, 1991, 245, SHORTT H.E., BARRAUD P.J. & CRAIGHEAD A.C. Note on a massive infection of the pharynx of Phlebotomus argentipes with Herpetomonas donovani. Indian Journal of Medical Research, 1926, 26, THEODOS C, RFBEIRO J.M.C. & TITUS R.G. Analysis of enhancing effect of sand fly saliva on Leishmania infection in mice. Infection and Immunity, 1991, 59, TITUS R.G. & RIBEIRO J.M.C. Salivary gland lysates from the sandflylutzomyia longipalpis enhance Leishmania infectivity. Science, 1988, 239, Reçu le 20 juin 1995 Accepté le 20 septembre